Aged nanoplastics reprogram the ER stress-autophagy crosstalk: A mechanistic gateway to skeletal malformations in zebrafish

Micro(nano)plastics (MNPs) are pervasive in aquatic environments, with aged MNPs being the predominant form due to environmental weathering. However, their developmental toxicity, particularly regarding skeletal formation, remains poorly understood. In this study, aged polystyrene nanoplastics (PSNPs) were prepared via 48-hour UV irradiation and compared with pristine PSNPs in zebrafish embryo exposures starting within 4 h post-fertilization (hpf) and lasting for 7 days. Both pristine and aged PSNPs induced skeletal malformations, but aged PSNPs caused more severe effects, including decreased hatching rates, increased mortality, and altered larval body length. Histopathological analysis revealed disrupted muscle fiber organization and lipid accumulation in the yolk sac. Elevated reactive oxygen species (ROS) and calcium ion levels were observed in both treatment groups. Aged PSNPs significantly upregulated GRP78 and RAB7 protein expression, increased transcription of the Endoplasmic Reticulum stress (ER stress) marker eif2α, and downregulated key genes involved in bone development (bmp2b, bmp4) and autophagy (atg13, atg5). These findings indicate that aged PSNPs exacerbate oxidative stress, activate ER stress pathways, inhibit autophagy, and impair skeletal development. This study highlights the heightened developmental toxicity of environmentally aged MNPs and underscores the necessity of including aged plastic particles in ecological risk assessments.